Ambient hygiene receives increasing attention in laboratories, doctors' receptions, at home, in public facilities and industrial production plants. The trend is towards developing methods with which the person using or cleaning a space can quickly ensure its hygiene. Such methods should be extremely simple, user-friendly, rapid and inexpensive.
Hygiene can be ensured by determining the presence of microbes, such as bacterial concentrations or substances promoting bacterial growth, on the surfaces. Analysing microbes from surfaces using current methods is slow and requires professional expertise. An analysis of substances—e.g., sugars and proteins—facilitating microbial e.g. bacterial or fungi growth indicates the cleanliness of surfaces with almost comparable reliability.
There are quick and sensitive tests available for determining the presence of protein that are based on the reaction of bromine cresol green with proteins. Such tests were described in, e.g., patent application WO 2006/122733, which exhaustively discusses existing test formats based on the use of various membranes. The application also extensively discusses various methods for applying reagents to the membrane as roll-to-roll or other printing techniques.
Most of the known methods of analysis of carbohydrates such as sugars are based on methods in which sugars are indicated by colour change. Most methods based on colour change were developed for the spectrophotometric assay of sugars. To date, there are no inexpensive, rapid and easy tests available to determine the presence of sugars on surfaces. A problem related to sugars is their stable structure, which means that their analysis requires the presence of specific and likely unstable enzymes, incubation at elevated high temperatures, prolonged reaction times and/or chemicals hazardous to health or unsafe to be used.
There are several methods available for analysing sugars that are based on colour change. Methods based on the reduction of copper include Fehling's reagents, arsenomolybdate and the BCA (bicinchoninic acid) assay. Other methods applicable to sugar determination include the iron cyanide and DNS (dinitrosalicylic acid) methods, methods based on acetal formation, the anthrone method, indication methods including phenazine group, Schiff's reagent and tetrazolium blue, as well as boronic sensors based on circular dichroism, photoabsorption and fluoresence. Other methods applicable to determining sugars include enzymatic methods such as glucose-oxidase/perodixase and hexokinase, and luminescence methods including bioluminescence and chemiluminescence.
The available methods as such are not applicable for use in a test format where sugars are detected using a fabric method. All of the methods based on copper reduction require heating in order to effect the reaction sufficiently rapidly.
In U.S. Pat. No. 6,586,195, Janus Green B is used to indicate sugars. The patent demonstrates that reducing sugars are capable of reducing Janus Green B in sufficiently high concentrations, in the order of 10 g/l, in basic conditions, in which case Janus Green B changes from blue to grey. The colour change is not optimal since this reaction—a change from blue to grey—makes it very difficult to determine the result of the test with concentrations close to the detection limit.
Methods based on acetal formation and the anthrone method use strong acids in high concentration, which makes these methods unsuitable in the development of a fabric-based rapid test.
Indicator methods involve a simple reagent composition, which reduces the required volume of reagents to be printed onto the fabric. With sufficiently high sugar concentrations, changes in indicator colour can also be observed even at room temperature. The drawback is that only sugars with high reduction power, e.g. fructose, can be detected. There is also a lack of commercially available indicators.
Enzymatic and luminescence-based methods are sensitive and quick. The drawbacks associated with some enzymes include their cost and their unstable characteristics. Furthermore, the specific action of enzymes i.e. they act only on particular sugars precludes the use of such enzymes in rapid tests, which should be able to indicate a total level of all or almost all carbohydrates. Luminescence-based methods are viable only in connection with sugar-modifying enzymes and thus share the same problems as enzymatic methods.
Well-known methods familiar to experts in the field and the reagents they employ are not as such applicable for quick diagnostic methods. For example, the iron cyanide method is not suitable for quick analysis of sugars since in an acidic environment cyanide is released as hydrogen cyanide, a highly toxic substance. Some methods may not work at room temperature (e.g., the DNS method or Janus Green B), or they may have poor stability (e.g., enzyme-requiring methods, Schiff's reagent and acetal-forming reagents). Several methods also require highly acidic or alkaline conditions.
Accordingly, there is a need for a rapid and sensitive test for determination of carbohydrates on surfaces. Especially there is a need for a test which can be performed without an increase in temperature.